The use of Kappa goniometers for crystal reorientations can be useful in a number of circumstances in MX (Brockhauser et al., 2011). These include the case where Bijvoet pairs of reflections (a reflection and the Friedel pair of its symmetry equivalent, e.g. hkl and hk¯l) can be measured on the same diffraction image by properly aligning an even-fold symmetry axis along the spindle. Hence, anomalous differences can be measured at the same time and radiation damage induced non-isomorphism within these Bijvoet pairs can be minimized, resulting in more accurate measurements of the anomalous differences. Aligning a specific symmetry axis can result in collecting a complete dataset within a reduced rotation range (Dauter, 1999) so that the total dose can be lowered leading to less severe radiation damage.  Another example of an advantageous crystal reorientation is the alignment of the densest axis in reciprocal space, usually corresponding to the longest unit cell axis. By aligning this axis parallel to the spindle, the overlap of spots can be minimized.

 

A simple workflow has been implemented in MXCuBE using a DAWN workflow to guide the user through the steps required to reorient a crystal. This is a three step, iterative protocol which contains the initial characterisation of the sample; the calculation of a set of preferred orientations; and testing the diffraction quality and predicting data collection statistics at different orientations until a satisfactory result is achieved. The implemented workflow provides a menu allowing the following re-orientation targets to be defined in EDNA. These allow users to choose the most appropriate strategy according to the needs of their samples:

 

The “Cell” option– aligns a reciprocal unit cell axis along the spindle. This option is useful for verifying crystal symmetries as well as investigating spot overlaps.

 

 “Anomalous” – aligns an even-fold symmetry axis along the spindle to measure the anomalous signal between the Bijvoet pairs on the same images.

 

The “Smart Spot Separation” option maximises spot separation while maintaining the highest possible completeness by avoiding the blind zones during data collection. This is achieved by a slight mis-alignment of the longest unit cell edge of the crystal when approaching the optimal orientation.

 

“Smallest Overall Oscillation” allows the– reorientation of the crystal, so the collection of a single sweep complete dataset would require the minimum overall oscillation.

 

The workflow is started by selecting "Kappa Reorientation" in the Advanced tab and adding it to the queue. A menu is then presented where the user selects the reorientation option (as explained above) required: cell, anomalous, smart spot separation or minimum oscillation:

 

mxcube_WF_114.png

 

The user then selects the characterisation required by EDNA, i.e number of images, angle between those images and the exposure time.  An EDNA characterisation is then launched and a proposed reorientation of the crystal is presented.  After accepting the reorientation the crystal is moved and the user must validate the centred position or re-centre the crystal. At this point another EDNA characterisation is launched. If the orientation is optimal for the requirements a data collection strategy is proposed, if a better orientation can be found a new reorientation is proposed. The final strategy can be edited, on clicking "continue" the strategy is sent to the queue and collected.